The long term goal of this project is to understand the mechanism by which active transport is carried out in the multi-subunit F0F1 ATP synthase. Recent observations have established that two or more of the subunits rotate relative to the others as a part of the catalytic mechanism. Amino acid replacements that cause inefficient coupling between transport and catalysis have been found to alter the energy of interaction between the rotating gamma subunit and catalytic beta subunits. In Specific Aim 1, random and site-directed mutations will be introduced to define the molecular features of the interfaces between subunits. Mutant enzymes will be assessed for perturbations in the transmission of energy between transport and catalysis of ATP synthesis. In Specific Aim 2, thorough kinetic and thermodynamic characterizations of mutant enzymes will be carried out and this data will be used to derive linear free energy and isokinetic relationships. From these analyses, developed from the characteristics of several mutant enzymes, the roles in the catalytic and coupling mechanisms of a specific amino acid or region are determined. In Specific Aim 3, a series of cysteine replacements will be introduced to probe the transport mechanism in the membranous f0 sector. There is little structural information about this portion of the complex. Nitroxide spin labels will be conjugated to the cysteine residues and electron paramagnetic resonance spectroscopy will be used to determine topology, and secondary and tertiary structural features. Focus will be on subunits a and c both of which are involved in transport. Furthermore the spin labels will be used to determine changes in molecular dynamics of the complex under the imposition of an electrochemical gradient of protons. These studies will seek to understand the molecular mechanisms which determine the efficiency of energy metabolism which is critical to all life processes. This is the most evident in genetic lesions of the F0F1 ATP synthase which effectively reduce its ability to produce ATP. These mutations affect cells with high metabolic rates, and in particular usually appear as neurodegenerative disorders.